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Abstract:

The present invention methods and systems for determining copy number
variation of a target polynucleotide in a genome of a subject including
amplification based techniques. Methods can include pre-amplification of
the sample followed by distribution of sample and a plurality of reaction
volumes, quantitative detection of a target polynucleotide and a
reference polynucleotide, and analysis so as to determine the relative
copy number of the target polynucleotide sequence in the genome of the
subject.

Claims:

1-17. (canceled)

18. A method for determining the relative copy number of a target
polynucleotide sequence in a genome of a subject, comprising:
pre-amplifying a target gene sequence and a reference gene sequence in a
sample containing human, non-human animal, plant, bacterial or fungal
genomic DNA of the subject such that multiple copies of said target gene
sequence that are linked together in the genome will be amplified
separately; assaying the target gene sequence and the reference gene
sequence of the preamplified sample by digital PCR; and determining a
ratio of (a) to (b), where (a) is the number of amplified polynucleotide
molecules containing the target gene sequence and (b) is the number of
amplified polynucleotide molecules containing the reference gene sequence
wherein the pre-amplifying comprises conducting at least 3 cycles of a
polymerase chain reaction (PCR) so as to amplify the target gene sequence
and reference gene sequence in substantially equal proportion.

19. The method of claim 19 wherein the target sequence is a sequence for
which deletion or duplication is associated with a phenotype of interest.

20. The method of claim 18 wherein the sample is from a non-human animal,
plant, bacteria or fungus.

21. The method of claim 21 wherein the sample is from a plant.

22. The method of claim 20 wherein i) the ratio of (a) to (b) is about
0.5 and there is a deletion of (a) on one chromosome ii) the ratio of (a)
to (b) is about 1.5 and there is a duplication of (a) on one chromosome.

23. The method of claim 20, wherein: the reference gene sequence has a
predetermined genomic copy number N; determining the number of amplified
polynucleotide molecules containing the target gene sequence comprises
determining the number of reaction volumes in which the target gene
sequence or subsequence thereof is present (a); determining the number of
amplified polynucleotide molecules containing the reference
polynucleotide sequence comprises determining the number of reaction
volumes in which the reference gene sequence or subsequence thereof is
present (b); and the relative copy number of the target polynucleotide in
the genome is approximately equal to the product of N multiplied by the
ratio (a)/(b).

24. The method of claim 20, wherein pre-amplifying a target gene sequence
and a reference gene sequence comprises combining the sample with a
composition comprising primers specific for the target gene sequence and
primers specific for reference gene sequence, and conducting a polymerase
chain reaction (PCR) assay so as to separately amplify target gene
sequences and reference gene sequences in substantially equal proportion.

25. The method of claim 18 wherein pre-amplifying a target gene sequence
and a reference gene sequence comprises from 4 to 10 cycles.

28. The method of claim 20 wherein pre-amplifying a target gene sequence
and a reference gene sequence comprises from 4 to 15 cycles.

29. The method of claim 23, wherein the reaction volumes are disposed in
a microfluidic device, and the first polynucleotide amplification is
conducted in a reaction volume separate from the microfluidic device.

30. The method of claim 23, wherein prior to assaying the target
polynucleotide sequence and the reference polynucleotide sequence of the
pre-amplified sample by digital PCR, all or a portion of the amplified
sample is combined with reagents selected for quantitative amplification
of target gene sequence and reference gene sequence.

31. The method of claim 30 wherein the primers used in the
pre-amplification step to amplify the reference gene sequence are the
same as those used in assaying the reference gene sequence of the
pre-amplified sample by digital PCR.

32. The method of claim 31 wherein the primers used in the
pre-amplification step to amplify the target gene sequence are the same
as those used in assaying the target gene sequence of the pre-amplified
sample by digital PCR.

33. The method of claim 23, wherein the reagents comprise a first probe
that selectively hybridizes to a target gene sequence and a second probe
that selectively hybridizes to a reference gene sequence under conditions
suitable for polynucleotide amplification.

34. The method of claim 33, wherein the first and second probes comprise
different detectable labels, and wherein binding of the first or second
probe or degradation of the first or second probe upon polymerase chain
reaction (PCR) based polymerization results in a change in detectable
fluorescence of the respective detectable label.

35. The method of claim 20, wherein a ratio of target gene sequence to
reference gene sequence substantially deviating from a value of 1
indicates an abnormal target gene sequence copy number in the genome.

36. The method of claim 20, wherein determining the relative copy number
of the target gene sequence comprises detecting a loss of heterozygosity
in the genome.

37. The method of claim 20, wherein a ratio of target gene sequence to
reference gene sequence with a value substantially greater than or less
than 1 indicates a loss of heterozygosity in the genome.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. §119(e) of
U.S. Provisional Patent Application No. 60/967,897, filed Sep. 7, 2007,
the full disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention relates to a method for determining copy number
variation within a genome from small populations or individuals and finds
application in biology and medicine.

BACKGROUND OF THE INVENTION

[0003] "Digital PCR" refers to a method in which individual nucleic acid
molecule present in a sample are distributed to many separate reaction
volumes (e.g., chambers or aliquots) prior to PCR amplification of one or
more target sequences. The concentration of individual molecules in the
sample is adjusted so that after distribution each reaction volume
contains fewer than one discrete polynucleotide molecule (or aggregate of
linked polynucleotide molecules), and most chambers contain zero or one
molecule. Amplification of a target sequence results in a binary digital
output in which each chamber is identified as either containing or not
containing the PCR product indicative of the presence of the
corresponding target sequence. A count of reaction volumes containing
detectable levels of PCR end-product is a direct measure of the absolute
nucleic acids quantity. In one version of Digital PCR, polynucleotide
molecules are distributed by partitioning them into separate reaction
volumes. One partition method uses the BioMark® 12.765 Digital Array
(Fluidigm Corp., South San Franscisco, Calif.). This chip utilizes
integrated channels and valves that partition mixtures of sample and
reagents into 765 nanolitre volume reaction chambers. DNA molecules in
each mixture are randomly partitioned into the 765 chambers of each
panel. The chip is then thermocycled and imaged on Fluidigm's BioMark
real-time PCR system and the positive chambers that originally contained
1 or more molecules can be counted by the digital array analysis
software. For discussions of Digital PCR see, for example, Vogelstein and
Kinzler, 1999, Proc Natl Acad Sci USA 96:9236-41; McBride et al., U.S.
Patent Application Publication No. 20050252773, especially Example 5;

[0004] Copy number variations (CNVs) are the gains or losses of genomic
regions which range from 500 bases on upwards in size (often between five
thousand and five million bases). Whole genome studies have revealed the
presence of large numbers of CNV regions in human and a broad range of
genetic diversity among the general population. CNVs have been the focus
of many recent studies because of their roles in human genetic disorders.
See, for example Iafrate et al., 2004, Detection of large-scale variation
in the human genome. Nat Genet 36: 949-951; Sebat et al., 2004,
Large-scale copy number polymorphism in the human genome. Science 305:
525-528; Redon et al., 2006, Global variation in copy number in the human
genome. Nature 444: 444-454; Wong et al., 2007, A comprehensive analysis
of common copy-number variations in the human genome. Am J Hum Genet 80:
91-104; Ropers, 2007, New perspectives for the elucidation of genetic
disorders. Am J Hum Genet 81: 199-207; Lupski, 2007, Genomic
rearrangements and sporadic disease. Nat Genet 39: S43-S47, each of which
is incorporated by reference. Aneuploidy, such as trisomy or whole
chromosome deletion, is a limiting type of copy number variation
associated with a variety of human diseases.

BRIEF SUMMARY OF THE INVENTION

[0005] The invention relates to a method for determining copy number
variation within a genome from small populations or individuals. The
method provides for the preamplification of the gene of interest in a
sample prior to analysis by digital PCR. The preamplification step allows
for the distribution of individual copies of the gene to be distributed
into individual PCR reaction samples for detection in a manner that is
more representative of actual copy number than when determined by digital
PCR without preamplification.

[0006] Digital PCR-based methods of the invention have the ability to
distinguish less than two-fold differences in gene copy number with great
accuracy. For example, it can differentiate between 1, 2, 3 and 4 copies
of genes in different samples. In order to ensure that apparent
difference in gene copy numbers in different samples are real, and not
distorted by differences in sample amounts, we use a term called relative
copy number. The relative copy number of a gene (per human genome) can be
expressed as the ratio of the copy number of a target gene to the copy
number of a single copy reference gene in a DNA sample, which is usually
1. For example, the RNaseP gene is a single-copy gene encoding the RNA
moiety for the RNaseP enzyme and may be used as the reference gene in a
copy number assay.

[0007] A commercially available digital array chip, such as that
illustrated in FIG. 3, for performing digital PCR, has been used to
quantitate DNA in a sample. The chip has 12 sample input ports for
introduction of a sample mixture. Each sample mixture is partitioned into
765 reaction chambers in each of the 12 panels. As is described in the
literature (see, e.g., McBride et al., U.S Patent Application Publication
No. 20050252773) the ability to quantitate DNA in samples is based on the
fact that, when an appropriate quantity of DNA is introduced, single DNA
molecules are randomly distributed in the chambers.

[0008] By using two assays for two genes (for example RNase P and another
gene of interest) with two fluorescent dyes on one chip, it is possible
to simultaneously quantitate both RNase P and the other gene in the same
DNA sample and obtain a good estimate of the ratio of these two genes and
the copy number of the gene of interest.

[0009] However, when duplicated, multiple copies of one gene might be
closely linked on the same chromosome and therefore can not be
partitioned from each other, even on the Digital Array. As a result,
multiple copies would behave as one molecule and the total number of
copies of the gene would be greatly underestimated.

[0010] The present invention addresses this problem by including a
preamplification step in the process. Preamplification is a PCR reaction
with primers for both the gene of interest and a reference gene (e.g.,
the RNase P gene). It is typically performed for a limited number of
thermal cycles (for example 10 cycles); assuming equal PCR efficiencies,
the copy numbers of both genes are proportionally increased in the
preamplification step. Using this process, even if multiple copies of a
gene are linked together on the genome, after preamplification, each copy
of the gene of interest will be amplified separately, and will be
partitioned separately into different chambers in the Digital Array.
Since the newly generated molecules of both genes reflect the original
ratio and they are not linked any more, a digital chip analysis can
quantitate the molecules of the two genes and measure the ratio of the
two genes (therefore the copy number of the gene of interest) accurately.

[0011] Thus, the present invention provides systems and related methods
for performing gene-based analyses. More specifically, the methods and
systems of the present invention generally relate to determining copy
number variation of a polynucleotide of interest in a sample from a
subject.

[0012] In one aspect the invention provides a method for determining the
relative copy number of a target polynucleotide sequence in a genome of a
subject, including the steps of:

[0013] a) pre-amplifying a target gene sequence and a reference gene
sequence in a sample containing genomic DNA of the subject; thereby
producing an amplified sample;

[0014] b) carrying out digital PCR by distributing product of (a) into a
plurality of isolated reaction volumes, amplifying target and reference
gene sequences in each reaction volume, and determining the relative
quantity of target and reference gene sequences in the amplified sample,
where the relative quantity of the target and reference gene sequences in
the amplified sample correspond to relative quantity of the target and
reference gene sequences in the genome.

[0015] In a related aspect the invention provides a method for determining
the relative copy number of a target polynucleotide sequence in a genome
of a subject, including the steps of:

[0016] pre-amplifying a target gene sequence and a reference gene sequence
in a sample containing genomic DNA of the subject;

[0017] assaying the target gene sequence and the reference gene sequence
of the preamplified sample by digital PCR;

[0018] determining (a) the number of amplified polynucleotide molecules
containing the target gene sequence and (b) the number of amplified
polynucleotide molecules containing the reference gene sequence and
determining the ratio of (a) to (b).

[0019] In a related aspect the invention provides a method for determining
a copy number of a target polynucleotide sequence in a genome of a
subject, including the steps of:

[0020] conducting a first polynucleotide amplification of a DNA sample
obtained from a subject, wherein both a target polynucleotide sequence
and a reference polynucleotide sequence, said reference sequence having a
predetermined genomic copy number N, are amplified, thereby producing an
amplified sample;

[0021] distributing all or a portion of the amplified sample into a
plurality of isolated reaction volumes;

[0022] in each reaction volume conducting a second polynucleotide
amplification in which the target polynucleotide sequence or a
subsequence thereof is amplified if present and the reference
polynucleotide sequence or a subsequent thereof is amplified if present;

[0023] determining the number of reaction volumes in which the target
polynucleotide sequence or subsequence thereof is present A and
determining (b) the number of reaction volumes in which the reference
polynucleotide sequence or subsequence thereof is present B; where the
copy number of the target polynucleotide in the genome is approximately
equal to (A)/(B)×N.

[0024] In some embodiments the sample is from a human. In particular
embodiments the ratio of (a) to (b) is about 0.5 and there is a deletion
of (a) on one chromosome, or the ratio of (a) to (b) is about 1.5 and
there is a duplication of (a) on one chromosome. In some embodiments a
ratio of target gene sequence to reference gene sequence substantially
deviating from a value of 1 indicates an abnormal target gene sequence
copy number in the genome of the patient.

[0025] In some embodiments conducting the first polynucleotide
amplification includes combining the biological sample with a composition
comprising primers specific for the target polynucleotide sequence and
primers specific for reference polynucleotide sequence, and conducting a
polymerase chain reaction (PCR) assay so as to separately amplify target
polynucleotide and reference polynucleotide in substantially equal
proportion.

[0026] In some embodiments the first polynucleotide amplification has from
4 to 15 thermocycles. In some embodiments the reaction volumes are
disposed in a microfluidic device, and the first polynucleotide
amplification is conducted in a reaction volume separate from the
microfluidic device.

[0027] In some embodiments prior to the step of distributing, all or a
portion of the amplified sample is combined with reagents selected for
amplification of target gene sequence and reference gene sequence.
Usually a portion is used, and the amplified sample may be diluted prior
to distribution of a portion to the reaction volumes. In some embodiments
the amplification is PCR amplification.

[0028] In some embodiments the reference gene sequence amplification
primers used in the first polynucleotide amplification step are the same
as those used in the second polynucleotide amplification step. In some
embodiments the target gene sequence amplification primers used in the
first polynucleotide amplification step are the same as those used in the
second polynucleotide amplification step. In some embodiments the
reagents comprise a first probe that selectively hybridizes to a target
gene sequence and a second probe that selectively hybridizes to a
reference gene sequence under conditions suitable for polynucleotide
amplification. In some embodiments the first and second probes comprise
different detectable labels, and wherein binding of the first or second
probe or degradation of the first or second probe upon polymerase chain
reaction (PCR) based polymerization results in a change in detectable
fluorescence of the respective detectable label.

[0029] In some embodiments the reference gene sequence comprises a
polynucleotide sequence at least partially encoding an RNaseP enzyme,
beta-actin or GAPDH. In some embodiments, determining the relative copy
number of the target gene sequence comprises detecting a loss of
heterozygosity in the genome of the subject. In some embodiments a ratio
of target gene sequence to reference gene sequence with a value
substantially greater than or less than 1 indicates a loss of
heterozygosity in the genome of the patient.

[0030] For a fuller understanding of the nature and advantages of the
present invention, reference should be had to the ensuing detailed
description taken in conjunction with the accompanying drawings. The
drawings represent embodiments of the present invention by way of
illustration. The invention is capable of modification in various
respects without departing from the invention. Accordingly, the
drawings/figures and description of these embodiments are illustrative in
nature, and not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031]FIG. 1 is a flow chart illustrating general steps of an inventive
method as described herein.

[0032] FIGS. 2A-2B illustrate exemplary channel designs of a microfluidic
device, according to embodiments of the present invention.

[0033]FIG. 3 is a simplified diagram of a microfluidic device, according
to an embodiment of the present invention.

[0036]FIG. 6 illustrates exemplary loss of heterozygosity results
performed using a microfluidic device.

[0037]FIG. 7 is a graph depicting detection of loss of heterozygosity,
according to an embodiment of the present invention.

[0038] FIG. 8 is a schematic showing the partial results of an imaginary
experiment in which the copy number of target sequence T is determined. A
64×64 matrix of reaction volumes is illustrated in which a target
sequence was amplified and detected using VIC labeled (yellow) probes and
a single copy reference sequence were amplified and detected using FAM
labeled (green) probes. 19 reaction volumes with yellow-labels and 12
reaction volumes with green-labels are detected, indicating a ratio of
approximately 1.5 (19/12=1.58≈1.5) indicating there are three
copies of the target sequence per diploid genome.

DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention methods and systems for determining copy
number of a target polynucleotide sequence in a genome of a patient,
including variations in copy number associated with genetic diseases. In
particular, methods and systems described herein can be used to detect
copy number variation of a target polynucleotide in the genome of a
patient using genomic material present within a sample derived from the
patient. Techniques of the present invention will typically employ
polynucleotide amplification based assays to determine the relative
number of copies of a target polynucleotide sequence and a reference
polynucleotide sequence in a sample. The genomic copy number is known for
the reference sequence. As such, target polynucleotide copy number can be
analyzed relative to the reference polynucleotide so as to determine the
relative copy number of the target polynucleotide. The target and/or
reference polynucleotide sequences are sometimes referred to as "genes."
However, it will be appreciated the term "gene" does not indicate the
sequence necessarily encodes a protein (or RNA).

[0040] Copy number detection and analysis techniques can make use of
certain high-throughput devices suitable for so called "digital analysis"
or "digital PCR", such as microfluidic devices including a large number
or high density of small volume reaction sites (e.g., nano-volume
reaction sites or reaction volumes). Accordingly, copy number variation
detection and analysis techniques of the present invention can include
distributing or partitioning a sample among hundreds to thousands of
reaction volumes disposed in a reaction/assay platform or microfluidic
device, including exemplary devices described herein.

[0041] The methods of the present invention include a pre-amplification
step in which DNA (e.g., genomic DNA) from a biological sample is
amplified using the polymerase chain reaction (PCR) or other quantitative
amplification techniques. Exemplary biological samples include cells
(including lysed cells and cell homoginates), serum, and biological
fluids. While the methods herein are described generally with respect to
human DNA (e.g., to determine copy number variation in the genome of a
human patient), it will be recognized that the methods can be
modified/applied to any sample having variations in amounts of genetic
material. For example, the methods can be used for genetic analysis of
animals, plants, bacteria and fungi, as well as for genetic analysis of
human subjects. Methods for collecting and processing biological samples
containing DNA are well known and need not be discussed here. For the
assays of the invention, DNA may be isolated from cells or biological
fluids, or the assay may be carried out using, for example, a cell lysate
containing DNA. Thus, as used herein, "a DNA sample" can refer to DNA,
especially genomic DNA, in purified, semi-purified or un purified form.
As used herein, a step of "obtaining a DNA sample from a subject" refers
simply to the fact that the DNA sample is the starting material for
subsequent analytical steps (e.g., the preamplification step). "Obtaining
a DNA sample" does not imply the act of, for example, collecting cells
from a subject, or isolating DNA, but may simply be a matter of obtaining
a tube containing precollected DNA.

[0042]FIG. 1 illustrates general steps for performing the methods
described herein. In one illustrative embodiment the steps of the method
involve providing a pre-amplification master mix comprising assay
primers, a suitable buffer system, nucleotides, and DNA polymerase enzyme
(such as a polymerase enzyme modified for "hot start" conditions), adding
genomic DNA to the pre-amplification master mix, pre-amplifying the
sequence(s) of interest and reference sequences, and assaying the
preamplified sequences by digital PCR analysis (either in an endpoint
assay or a real time assay), and comparing the frequency of the target
sequence(s) relative to the frequency of the reference sequence. It will
be recognized that FIG. 1 is provided to aid in understanding the
invention, but is not intended to limit the invention.

[0043] In the initial step in FIG. 1, preamplification, a first
polynucleotide amplification of a DNA sample obtained from a subject is
carried out. In the preamplification step both a target polynucleotide
sequence and a reference polynucleotide sequence are amplified. Methods
for PCR amplification are well known and need to be described here.

[0044] In some embodiments, the target sequence is a sequence for which
deletion or duplication is associated with a phenotype of interest. In
some embodiments, the target sequence is a sequence for which deletion or
duplication is not associated with a known phenotype of interest, but for
which information about the distribution or correlation of the variation
in particular populations is desired.

[0045] The reference sequence is a sequence having a known (or assumed)
genomic copy number. Thus a reference sequence is one that is not likely
to be amplified or deleted in a genome. It is not necessary to
emperically determine the copy number of the reference sequence in each
assay. Rather, the copy number may be assumed based on the normal copy
number in the organism of interest. For example, one useful reference
sequence in the human genome is the sequence of the RNaseP gene, a
single-copy gene present in two copies per diploid genome (and having a
copy number of 1 per haploid genome). For illustration, other useful
reference sequences include β-actin and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH); however, it will be appreciated the invention is
not limited to a particular reference sequence.

[0046] Pre-amplification can be performed as a PCR reaction with primers
for both RNaseP (the reference gene) and the target gene of interest.
Typically, reactions are performed for a limited number of thermal cycles
(e.g., 5 cycles, or 10 cycles). In some embodiments, the optimal number
of cycles will depend on the PCR efficiencies for the reference gene and
target gene. In certain embodiments, the number of thermal cycles during
a pre-amplification assay can range from about 4 to 15 thermal cycles, or
about 4-10 thermal cycles. In certain embodiments the number of thermal
cycles can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more than
15.

[0047] Pre-amplification reactions preferably are quantitative or
proportional. That is, the relative number (ratio) of amplicons of the
target and reference sequences should reflect the relative number (ratio)
of target and reference sequence in the genomic (or other) DNA being
amplified. Methods for quantitative amplification are known in the art.
See, e.g., Arya et al., 2005, Basic principles of real-time quantitative
PCR, Expert Rev Mol Diagn. 5(2):209-19. In the case of duplicated genes,
primers should be selected such that each duplicated copy of the target
gene of interest is separately amplified. Thus, following selective
pre-amplification and distribution of the sample into separate reaction
volumes, a proportional number of amplicons corresponding to each
sequence will be distributed into the reaction volumes. Because the newly
generated molecules of both genes reflect the original ratio, a
subsequent copy number analysis can quantitate the number of molecules of
the target gene and the reference gene. As a result, the ratio of the two
genes can be measured accurately. Because the copy number of the
reference sequence is known, the copy number of the sequence of interest
can be determined.

[0048] It is desirable that the amplification efficiencies target and
reference sequences be similar or approximately equal, in order not to
introduce any bias in the ratio of the two gene copy numbers. For this
reason, primer pairs and amplification conditions should be selected to
obtain this result. The amplification efficiency of any pair of primers
can be easily determined using routine techniques (see e.g., Furtado et
al., "Application of real-time quantitative PCR in the analysis of gene
expression." DNA amplification: Current Technologies and Applications.
Wymondham, Norfolk, UK: Horizon Bioscience p. 131-145 (2004))

[0049] Although it is desirable that the amplification efficiencies target
and reference sequences be approximately equal, the limited number of
preamplification thermal cycles (typically less than 15, usually 10 or
less than 10, most often about 5) greatly mitigates any differences in
efficiency, such that the usual differences are likely to have an
insignificant effect on our results.

[0050] As noted, amplification methods are known in the art. For
illustration, the reaction mixture used for the pre amplification method
(pre-amplification composition or mix) typically contains an appropriate
buffer, a source of magnesium ions (Mg2+) in the range of about 1 to
about 10 mM, preferably in the range of about 2 to about 8 mM,
nucleotides, and optionally, detergents, and stabilizers. An example of
one suitable buffer is TRIS buffer at a concentration of about 5 mM to
about 85 mM, with a concentration of 10 mM to 30 mM preferred. In one
embodiment, the TRIS buffer concentration is 20 mM in the reaction mix
double strength (2×) form. The reaction mix can have a pH range of
from about 7.5 to about 9.0, with a pH range of about 8.0 to about 8.5 as
typical. Concentration of nucleotides can be in the range of about 25 mM
to about 1000 mM, typically in the range of about 100 mM to about 800 mM.
Examples of dNTP concentrations are 100, 200, 300, 400, 500, 600, 700,
and 800 mM. Detergents such as Tween® 20, Triton® X 100, and
Nonidet® P40 may also be included in the reaction mixture. Stabilizing
agents such as dithiothreitol (DTT, Cleland's reagent) or mercaptoethanol
may also be included. The pre-amplification reaction mix will contain
primers for the pre-amplification reaction. The primers are generally the
same sequence as those to be used in the subsequent PCR assays for which
the sample is being prepared although generally in reduced concentration.
The primer concentration can be greater, equal to, or less than the
primer concentrations used in the PCR assay. Embodiments include the use
of primers that are about 50, 25, 20, 10 or 5 times greater, equal to, or
10, 20, 35, 50, 65, 75, 100, 125, 150, 175, and 200 times less than that
of the primer concentration in the PCR assay. Primers used in the
pre-amplification can include random primers, poly A tails, and specific
primers designed for the PCR assay of interest.

[0051] The reaction mix can optionally contain a reference dye for
normalizing subsequent real quantitative PCR analysis results. An example
of a common commercially available reference dye is ROX. A commercially
available reaction mix containing ROX dye is CellsDirect 2×
Reaction Mix, Cat. Nos. 11754-100 and 11754-500, available from
Invitrogen Corporation.

[0052] A DNA polymerase enzyme (e.g., a Taq polymerase) is also added to
the reaction mix. In one embodiment a Taq polymerase such as
Platinum® Taq DNA is a recombinant Taq DNA polymerase complexed with
an antibody that inhibits polymerase activity at ambient temperatures.
Full polymerase activity is restored after the denaturation step in PCR,
providing a "hot start."

[0053] The pre-amplified samples prepared by the methods of the present
invention are particularly suited for digital PCR analyses and for
distinguishing chromosomal duplication of genes. In particular, a
pre-amplified sample is assayed in a plurality of low volume PCR
experiments. In digital PCR, identical (or substantially similar) assays
are run on a sample of the genomic DNA. The number of individual
reactions for a given genomic sample may vary from about 2 to over
1,000,000. Preferably, the number of assays performed on a sample is 100
or greater, more preferably, 200 or greater, more preferably, 300 or
greater. Larger scale digital PCR can also be performed in which the
number of assays performed on a sample is 500 or greater, 700 or greater,
765 or greater, 1,000 or greater, 2,500 or greater 5,000 or greater 7,500
or greater, or 10,000 or greater. The number of assays performed may also
be significantly large such up to about 25,000, up to about 50,000, up to
about 75,000, up to about 100,000, up to about 250,000, up to about
500,000, up to about 750,000, up to about 1,000,000, or greater than
1,000,000 assays per genomic sample. The quantity of DNA used in a
digital PCR assay is generally selected such that one nucleic acid
fragment or less is present in each individual digital PCR reaction.

[0054] As illustrated in FIG. 1, following the pre-amplification step, the
sample (or a portion thereof) comprising pre-amplification product having
proportionately amplified genetic material (e.g., amplicons corresponding
to target and reference polynucleotide sequences) is distributed into
discrete locations or reaction volumes such that each reaction well
includes, for example, an average of no more than about one amplicon per
volume. Thus, most reaction volumes will have no amplicon, one target
sequence amplicon, or one reference sequence amplicon. Generally it is
useful to dilute the preamplified sample (typically 1:10-1:20) and/or use
a small portion of the amplified sample so as to adjust the concentration
of amplicons so that only (on average) there are zero or one amplicons
per reaction volume. Although in some cases the product of the
pre-amplification step can be used without addition of further
amplification reagents (e.g., polymerase), it is generally useful to add
new reagents for the amplification including, optionally, different
primers. Thus, the biological sample, either prior to distribution or
after, can be combined with reagents selected for quantitative or
nonquantitative amplification of both a target polynucleotide sequence
and a reference polynucleotide 12 (Step 2).

[0055] Moreover, although the preamplification step is generally a
PCR-type amplification, the second amplification (i.e., amplification of
the amplicon sequences produced in the preamplification) can be carried
out using any amplification method such as; for example and not
limitation, Nasba (Compton, 1991. Nucleic Acid Sequence-based
Amplification, Nature 350: 91-91, 1991) and the Eberwine protocol (Van
Gelder et al., Amplified RNA synthesized from limited quantities of
heterogeneous cDNA. Proc Natl Acad Sci USA. 1990).

[0056] As noted above, it will be appreciated that the quantity of DNA
templates and amplicons (a function of the amount of starting genomic
DNA, the number of amplification cycles, the efficiency of amplification
and the size of the reaction volumes) will be adjusted to achieve the
desired distribution. One of skill in the art can determine the
concentration of amplicons in the pre-amplification products and
calculate an appropriate amount for input. More conveniently a set of
serial dilutions of the preamplification product can be tested. For
example, the device shown in FIG. 3 (commercially available from Fluidigm
Corp. as the BioMark 12.765 Digital Array) allows 12 dilutions to be
tested simultaneously. Optionally the optimal dilution can be determined
by generating a linear regression plot. For the optimal dilution the line
should be straight and pass through the origin. Subsequently the
concentration of the original samples can be calculated from the plot.

[0057] Following distribution, the genomic material contained within a
plurality of reaction chambers can be amplified to further conduct sample
assays so as to determine the number of reaction volumes in which the
amplicons corresponding to the target or reference sequence were
sequestered (FIG. 1, 14). The second amplification can be carried out
using the same primers as used in the preamplification or different
primers (e.g., a nested set).

[0058] Differential detection and analysis of the sample can be conducted
so as to distinguish signal stemming from the target polynucleotide
compared to the reference polynucleotide (FIG. 1, 16). For example,
analysis of separate reaction sites can be used to calculate the ratio of
the number of reaction volumes containing target polynucleotide sequences
and the number reaction volumes containing reference polynucleotide
sequences. Methods can further include detecting and analyzing
genetically-related information about target sequences in the genome of a
subject, including detection of genetic deletions or duplications, loss
of heterozygosity, and the like, such as aneuploidy (e.g. trisomy) and
numerous other genetic abnormalities. Further detail on method steps,
including various differential detection and analysis techniques, is
provided below.

[0059] As disclosed above, sample containing pre-amplification product or
non-amplified genetic material can be distributed into discrete locations
or reaction volumes of a detection and analysis platform. Distribution of
the sample can be performed using a variety of techniques and devices
such as, for example, flow-based distribution in microfluidic devices
including a plurality of small volume reaction sites/chambers. Generally,
the distribution step of the methods described herein are implemented to
isolate sample material of interest, e.g., target and reference sequences
into individual reaction sites for later detection and analysis.

[0060] Within each of a plurality of reaction sites or volumes, one or
more amplification assays can be conducted, including multiplex reactions
detection quantitative analysis/amplification of target polynucleotide
sequence and a selected reference polynucleotide sequence. The ratio of
detected sequences in a sample can be calculated using detection
techniques such as digital PCR analysis, monitoring real-time PCR curves
and/or comparing end point images of positive reaction chambers for one
assay versus another assay. Alternatively, the concentration of any
sequence in a DNA sample (copies/μL) can be calculated using the
number of positive reaction chambers in the device that contain at least
one copy of that sequence and a ratio of concentrations of target and
reference sequences can be determined to calculate copy number. See
copending U.S. patent application Ser. No. 12/170,414, "Method and
Apparatus for Determining Copy Number Variation Using Digital PCR," which
is incorporated by reference for all purposes. Also see Dube et al.,
2008, "Mathematical Analysis of Copy Number Variation in a DNA Sample
Using Digital PCR on a Nanofluidic Device" PLoS ONE 3(8): e2876.
doi:10.1371/journal.pone.0002876, which is incorporated by reference for
all purposes.

[0061] As described above, the present invention includes methods and
amplification based techniques for determining copy number variation of a
target polynucleotide, e.g., in a genome of a patient, and in some
instances, a pre-amplification step can be performed prior to
distribution of the sample in a microfluidic device for subsequent
quantitative amplification and analysis. Pre-amplification may be
desired, for example, where multiple copies of one target gene are
closely spaced on the same chromosome, and thus the target sequences
cannot be optimally partitioned from each other during quantitative
analysis, e.g., as distributed in the microfluidic device. In such cases,
multiple copies of the target gene may be under-counted or quantitated as
one molecule rather than two. Accordingly, the total number of copies of
the gene can be underestimated.

[0062] According to the present invention, CNV calculations will typically
include calculation of "relative copy number" so as to advantageously
distinguish apparent differences in gene copy numbers in different
samples from distortion or assay noise/error, such as distortion caused
by differences in sample amounts. The relative copy number of a gene (per
genome) can be expressed as the ratio of the copy number of a target gene
to the copy number of a single copy reference gene in a DNA sample of
known concentration (copy number) in the genome of the patient, which is
typically equal to 1. By using two assays for the two genes (the target
polynucleotide and the reference polynucleotide) with two different
labels (e.g., fluorescent dyes) on the same digital array, the methods
described herein can be used to simultaneously quantitate both genes in
the same DNA sample. Alternatively, and less conveniently, the target
amplicons (from preamplification) can be amplified on one chip of set of
reaction volumes and the test amplicons (from preamplification) can be
assayed in a different set of amplicons and the data compared. The ratio
of these two genes is the relative copy number of the target
polynucleotide sequence, or gene of interest, in a DNA sample. In one
approach this method can be summarized as determining the number of
reaction volumes in which the target polynucleotide sequence or
subsequence thereof is present (A) and determining the number of reaction
volumes in which the reference polynucleotide sequence or subsequence
thereof is present (B), and determining that the copy number of the
target polynucleotide in the genome is approximately equal to
(A)/(B)×N, where N is the predetermined genomic copy number of the
reference sequence. It will be understood that the (A)/(B)×N is
related approximately to copy number because ploidy in most organisms are
low (e.g., humans normally have two copies of somatic chromosomes) while
the number of amplicons detected in the present invention is inherently
subject to experimental error. For example, (A) may be experimentally
determined to be 936 and (B) may be experimentally determined to be 596
and N may be 1 per haploid genome. (A)/(B)×N is equal to 1.57
(approximately 1.5) which would be understood to indicate that be
approximately 1.5 copies of A per haploid genome (i.e., trisomy of A).
See FIG. 8 and Example below.

[0063] A variety of detection platforms or microfluidic devices and
methods can be used in the practice of the invention. In some embodiments
devices can be constructed using a wide variety of materials, such as
glass, plastic, silicon, elastomeric polymers (e.g.,
polydimethylsiloxane, polyurethane, or other polymers). In certain
embodiments of the present invention, microfluidic devices used to carry
out aspects of the present invention are typically constructed at least
in part from elastomeric materials and constructed by single and
multilayer soft lithography (MSL) techniques and/or sacrificial-layer
encapsulation methods (see, e.g., Unger et al., 2000, Science
288:113-116, and PCT Publication WO 01/01025, both of which are
incorporated by reference herein in their entirety for all purposes).
Utilizing such methods, microfluidic devices can be designed in which
solution flow through flow channels of the device is controlled, at least
in part, with one or more control channels that are separated from the
flow channel by an elastomeric membrane or segment. This membrane or
segment can be deflected into or retracted from the flow channel with
which a control channel is associated by applying an actuation force to
the control channels. By controlling the degree to which the membrane is
deflected into or retracted out from the flow channel, solution flow can
be slowed or entirely blocked through the flow channel. Using
combinations of control and flow channels of this type, one can prepare a
variety of different types of valves and pumps for regulating solution
flow as described in extensive detail in Unger et al., supra, PCT
Publication WO 02/43615 and WO 01/01025.

[0064] Sample distribution in the microfluidic devices described herein
can be implemented in-part due to certain properties of elastomeric
materials, which are recognized generally in the art. For example,
Allcock et al. (Contemporary Polymer Chemistry, 2nd Ed.) describes
"elastomers" or "elastomeric material" in general as polymers existing at
a temperature between their glass transition temperature and liquefaction
temperature. Elastomeric materials exhibit elastic properties because the
polymer chains readily undergo torsional motion to permit uncoiling of
the backbone chains in response to a force, with the backbone chains
recoiling to assume the prior shape in the absence of the force. In
general, elastomers deform when force is applied, but then return to
their original shape when the force is removed. The elasticity exhibited
by elastomeric materials can be characterized by a Young's modulus. The
elastomeric materials utilized in the microfluidic devices disclosed
herein typically have a Young's modulus of between about 1 Pa-1 TPa, in
other instances between about 10 Pa-100 GPa, in still other instances
between about 20 Pa-1 GPa, in yet other instances between about 50 Pa-10
MPa, and in certain instances between about 100 Pa-1 MPa. Elastomeric
materials having a Young's modulus outside of these ranges can also be
utilized depending upon the needs of a particular application.

[0065] Given the tremendous diversity of polymer chemistries, precursors,
synthetic methods, reaction conditions, and potential additives, a wide
range of properties can be selected for certain uses and applications.
Therefore, with regards to the present invention, there are a large
number of possible elastomer systems that can be used to make monolithic
elastomeric microvalves and pumps. Some of the microfluidic devices
described herein are fabricated from an elastomeric polymer such as GE
RTV 615 (formulation), a vinyl-silane crosslinked (type) silicone
elastomer (family). However, the present microfluidic systems are not
limited to this one formulation, type or even this family of polymer;
rather, nearly any elastomeric polymer is suitable. The choice of
materials typically depends upon the particular material properties
(e.g., solvent resistance, stiffness, gas permeability, and/or
temperature stability) required for the application being conducted.
Additional details regarding the type of elastomeric materials that can
be used in the manufacture of the components of the microfluidic devices
disclosed herein are set forth in Unger et al. (2000) Science
288:113-116, and PCT Publications WO 02/43615, and WO 01/01025, and which
are incorporated herein by reference in their entirety for all purposes.

[0066] Device Fabrication and Thermocycling.

[0067] As indicated, techniques of the present invention can incorporate
use of a wide variety of detection platforms, including high throughput
microfluidic devices suitable for digital analysis or digital PCR.
Aspects of device fabrication, system components, and thermocyling
aspects are described in greater detail below.

[0068] In one embodiment, microfluidic devices suitable for use in the
present invention can be constructed utilizing single and multilayer soft
lithography (MSL) techniques and/or sacrificial-layer encapsulation
methods. One basic MSL approach involves casting a series of elastomeric
layers on a micro-machined mold, removing the layers from the mold and
then fusing the layers together. In the sacrificial-layer encapsulation
approach, patterns of photoresist are deposited wherever a channel is
desired. These techniques and their use in producing microfluidic devices
is discussed in detail, for example, by Unger et al. (2000) Science
288:113-116, and by Chou, et al. (2000) "Integrated Elastomer Fluidic
Lab-on-a-chip-Surface Patterning and DNA Diagnostics," in Proceedings of
the Solid State Actuator and Sensor Workshop, Hilton Head, S.C.; and in
PCT Publication WO 01/01025, each of which is incorporated herein by
reference in their entirety for all purposes.

[0069] In brief, the foregoing exemplary fabrication methods initially
involve fabricating mother molds for top layers (e.g., the elastomeric
layer with the control channels) and bottom layers (e.g., the elastomeric
layer with the flow channels) on silicon wafers by photolithography with
photoresist (Shipley SJR 5740). Channel heights can be controlled
precisely by the spin coating rate. Photoresist channels are formed by
exposing the photoresist to UV light followed by development. Heat reflow
process and protection treatment is typically achieved as described by M.
A. Unger, H.-P. Chou, T. Throsen, A. Scherer and S. R. Quake, Science
(2000) 288:113, which is incorporated herein by reference in its
entirety. A mixed two-part-silicone elastomer (GE RTV 615) is then spun
into the bottom mold and poured onto the top mold, respectively. Spin
coating can be utilized to control the thickness of bottom polymeric
fluid layer. The partially cured top layer is peeled off from its mold
after baking in the oven at 80° C. for 25 minutes, aligned and
assembled with the bottom layer. A 1.5-hour final bake at 80° C.
is used to bind these two layers irreversibly. Once peeled off from the
bottom silicon mother mold, this RTV device is typically treated with HCL
(0.1N, 30 min at 80° C.). This treatment acts to cleave some of
the Si--O--Si bonds, thereby exposing hydroxy groups that make the
channels more hydrophilic.

[0070] The device can then optionally be hermetically sealed to a support.
The support can be manufactured of essentially any material, although the
surface should be flat to ensure a good seal, as the seal formed is
primarily due to adhesive forces. Examples of suitable supports include
glass, plastics and the like.

[0071] The devices formed according to the foregoing method result in the
substrate (e.g., glass slide) forming one wall of the flow channel.
Alternatively, the device once removed from the mother mold is sealed to
a thin elastomeric membrane such that the flow channel is totally
enclosed in elastomeric material. The resulting elastomeric device can
then optionally be joined to a substrate support.

[0072] Layer Formation

[0073] In one embodiment, microfluidic devices, including those in which
reagents are deposited at the reaction sites during manufacture, are
formed of three layers. The bottom layer is the layer upon which reagents
are deposited. The bottom layer can be formed from various elastomeric
materials as described in the references cited above on MLS methods.
Typically, the material is polydimethylsiloxane (PDMS) elastomer. Based
upon the arrangement and location of the reaction sites that is desired
for the particular device, one can determine the locations on the bottom
layer at which the appropriate reagents should be spotted. Because PDMS
is hydrophobic, the deposited aqueous spot shrinks to form a very small
spot. The optionally deposited reagents are deposited such that a
covalent bond is not formed between the reagent and the surface of the
elastomer because, as described earlier, the reagents are intended to
dissolve in the sample solution once it is introduced into the reaction
site.

[0074] The other two layers of the device are the layer in which the flow
channels are formed and the layer in which the control and optionally
guard channels are formed. These two layers are prepared according to the
general methods set forth earlier in this section. The resulting two
layer structure is then placed on top of the first layer onto which the
reagents have been deposited. A specific example of the composition of
the three layers is as follows (ration of component A to component B):
first layer (sample layer) 30:1 (by weight); second layer (flow channel
layer) 30:1; and third layer (control layer) 4:1. It is anticipated,
however, that other compositions and ratios of the elastomeric components
can be utilized as well. During this process, the reaction sites are
aligned with the deposited reagents such that the reagents are positioned
within the appropriate reaction site.

[0075] In accordance with the present invention, thermocycling can be
performed on the microfluidic devices. In particular, thermocycling can
be used to run amplification reactions that facilitate analysis of sample
distributed within the reaction chambers.

[0076] A number of different options of varying sophistication are
available for controlling temperature within selected regions of the
microfluidic device or the entire device. Thus, as used herein, the term
temperature controller is meant broadly to refer to a device or element
that can regulate temperature of the entire microfluidic device or within
a portion of the microfluidic device (e.g., within a particular
temperature region or at one or more junctions in a matrix of blind
channel-type microfluidic device).

[0077] Generally, the devices are placed on a thermal cycling plate to
thermal cycle the device. A variety of such plates are readily available
from commercial sources, including for example the ThermoHybaid P×2
(Franklin, M A), MJ Research PTC-200 (South San Francisco, Calif.),
Eppendorf Part #E5331 (Westbury, N.Y.), Techne Part #205330 (Princeton,
N.J.).

[0078] To ensure the accuracy of thermal cycling steps, in certain devices
it is useful to incorporate sensors detecting temperature at various
regions of the device. One structure for detecting temperature is a
thermocouple. Such a thermocouple could be created as thin film wires
patterned on the underlying substrate material, or as wires incorporated
directly into the microfabricated elastomer material itself.

[0079] Various means of temperature detection/monitoring can be included
in a system/device of the present invention. For example, temperature can
also be sensed through a change in electrical resistance.
Thermo-chromatic materials are another type of structure available to
detect temperature on regions of an amplification device. Another
approach to detecting temperature is through the use of an infrared
camera. Yet another approach to temperature detection is through the use
of pyroelectric sensors. Other electrical phenomena, such as capacitance
and inductance, can be exploited to detect temperature in accordance with
embodiments of the present invention. Using known equations for thermal
diffusivity and appropriate values for the elastomers and glass utilized
in the device, one can calculate the time required for the temperature
within the reaction site to reach the temperature the controller seeks to
maintain.

[0080] In addition to the various potentially suitable material
compositions and properties, suitable microfluidic devices for use in the
present invention can include a variety of features, designs, channel
architectures, and the like. Devices will typically include a plurality
of "flow channels," which refer generally to a flow path through which a
solution can flow. Additionally, the devices can include "control
channels," or channels designed to interface with flow channels such that
they may be used to actuate flow within the flow channels. Devices can
further include features to further regulate fluid flow, such as a
"valve," which can include a configuration in which a flow channel and a
control channel intersect and are separated by an elastomeric membrane
that can be deflected into or retracted from the flow channel in response
to an actuation force. Also, certain embodiments may include a "via,"
which refers to a channel formed in an elastomeric device to provide
fluid access between an external port of the device and one or more flow
channels. Thus, a via can serve as a sample input or output, for example.

[0081] Numerous types of channel architectures or designs can be
implemented in the present invention. As illustrated in FIG. 2A, one type
of channel design that can be included in a device of the present
invention includes an open channel design. "Open channels" or "open-end
channels" refer to a flow channel disposed between separate via, such
that the flow channel has a entrance (e.g., inlet) separate from an exit
(e.g., outlet). In general, an open channel network design includes at
least two opposing flow channel via or inlets, which can be connected
about one or a plurality of branch flow channels to form an open channel
network. One or more valves formed by an adjacent/overlaying control
channel can be actuated to isolate discrete regions of the branch
channels to form reaction sites. Such valves provide a mechanism for
switchably isolating a plurality of reaction sites. As described herein,
devices can include one or more open flow channels from which one or more
channels branch. One or more reaction regions or reaction sites can be
disposed anywhere along a length of a flow channel. A valve formed by an
overlaying flow channel can be actuated to isolate the reaction site(s)
disposed along the channel, thereby providing a mechanism for switchably
isolating the reaction sites. Thus, each device can include a large
number of reaction sites (e.g., 10,000+) and can achieve high reaction
site densities, thereby allowing a significant reduction in the size of
these devices compared to traditional microfluidic devices. Open channel
designs can, for example, have branch flow channels that can be addressed
from more than one location/via. This design aspect may be particularly
advantageous, for example, if a particular channel/branch flow channel is
obstructed or blocked (e.g., due to manufacturing variation, defect,
etc.), as fluid can be entered from different directions and fill a
channel up to opposing sides of a particular blockage or obstruction. In
contrast, a channel accessible from only a single end having a blockage
may only be filled up to the point of the blockage or obstruction and, if
reaction sites exist beyond the blockage, those sites can be rendered
unusable.

[0082] As depicted in FIG. 2B, microfluidic devices suitable for use
according to the present invention may utilize a "blind channel" or
"blind fill" design. Such devices are characterized in part by having one
or more blind channels, or flow channels having a dead end or isolated
end such that solution can only enter and exit the blind channel at one
end (i.e., there is not a separate inlet and outlet for the blind
channel). These devices require only a single valve for each blind
channel to isolate a region of the blind channel to form an isolated
reaction site. During manufacture of this type of device, one or more
reagents for conducting an analysis can optionally be deposited at the
reaction sites, thereby resulting in a significant reduction in the
number of input and outputs. Thus, a flow channel network in
communication with the blind channels can be configured such that many
reaction sites can be filled with a single or a limited number of inlets
(e.g., less than 5 or less than 10). The ability to fill a blind flow
channel is made possible because the devices are made from elastomeric
material sufficiently porous such that air within the flow channels and
blind channels can escape through these pores as solution is introduced
into the channels. The lack of porosity of materials utilized in other
microfluidic devices precludes use of the blind channel design because
air in a blind channel has no way to escape as solution is injected.

[0083] In yet another embodiment, microfluidic devices of the present
invention can further optionally include guard channels in addition to
flow channels and valve or control channels. In order to reduce
evaporation of sample and reagents from the elastomeric microfluidic
devices that are provided herein, a plurality of guard channels can be
formed in the devices. The guard channels are similar to the control
channels in that typically they are formed in a layer of elastomer that
overlays the flow channels and/or reaction site. Hence, like control
channels, the guard channels are separated from the underlying flow
channels and/or reaction sites by a membrane or segment of elastomeric
material. Unlike control channels, however, the guard channels are
considerably smaller in cross-sectional area. In general, a membrane with
smaller area will deflect less than a membrane with larger area under the
same applied pressure. The guard channels are designed to be pressurized
to allow solution (typically water) to be flowed into the guard channel.
Water vapor originating from the guard channel can diffuse into the pores
of the elastomer adjacent a flow channel or reaction site, thus
increasing the water vapor concentration adjacent the flow channel or
reaction site and reducing evaporation of solution therefrom. For further
discussion of guard channels disposed in microfluidic devices and
suitable for use according to the present invention, see, McBride et al.,
U.S Patent Application Publication No. 20050252773, which is incorporated
herein by reference in its entirety for all purposes.

[0084] The devices further include a plurality of reaction sites, or
reaction volumes, at which reagents are allowed to react, and a device
may incorporate various means (e.g., pumps and valves) to selectively
isolate reaction sites. The reaction sites can be located at any of a
number of different locations within the device.

[0085] Because devices can include elastomeric materials that are
relatively optically transparent, reactions can be readily monitored
using a variety of different detection systems at essentially any
location on the microfluidic device. When MSL-type devices are used most
typically detection occurs at the reaction site itself. The fact that
such devices are manufactured from substantially transparent materials
also means that certain detection systems can be utilized with the
current devices that are not usable with traditional silicon-based
microfluidic devices. Detection can be achieved using detectors that are
incorporated into the device or that are separate from the device but
aligned with the region of the device to be detected.

[0086] In certain embodiments of the present invention, reactions within
the reaction volumes are conducted using mixes or reagents that are first
mixed (e.g., mixed with sample) in solution separate from the from the
chip and other system components and then introduced in solution.

[0087] Devices will typically be designed and configured to conduct
temperature controlled reactions, such as thermocycling amplification
reactions. Thus, a device can be configured/designed for use in
temperature control reactions (e.g., thermocycling reactions) within
reaction volumes. A device or portion thereof, e.g., the elastomeric
device, can be fixed to a support (e.g., a glass slide). The resulting
structure can then be placed on a temperature control plate, for example,
to control the temperature at the various reaction sites. In the case of
thermocycling reactions, the device can be placed on any of a number of
thermocycling plates.

[0088] As illustrated above, optional use of microfluidic devices to
implement the methods of the present invention can be conducted using a
wide variety of device features and designs. The following description
describes in greater detail exemplary configurations that can be utilized
to conduct a variety of analyses, including analyses requiring
temperature control (e.g., nucleic acid amplification reactions). It
should be understood, however, that these configurations are exemplary
and that modifications of these systems will be apparent to those skilled
in the art.

[0089]FIG. 3 is a simplified diagram of a microfluidic device, according
to an exemplary embodiment of the present invention. As illustrated in
FIG. 3, the microfluidic device, also referred to as a digital array, can
include a carrier 20, which can be made from materials providing suitable
mechanical support for the various elements of the microfluidic device.
As an example, the device is made using an elastomeric polymer. The outer
portion of the device has the same footprint as a standard 384-well
microplate and enables stand-alone valve operation. As described below,
there are 12 input ports corresponding to 12 separate sample inputs to
the device. The device can have 12 panels 22 and each of the 12 panels
can contain 765 6 nL reaction chambers with a total volume of 4.59 μL
per panel. Microfluidic channels 24 can connect the various reaction
chambers on the panels to fluid sources as described more fully below.

[0090] Pressure can be applied to an accumulator 26 in order to open and
close valves connecting the reaction chambers to fluid sources. As
illustrated in FIG. 3, 12 inlets 28 can be provided for loading of the
sample reagent mixture. 48 inlets 28 are used in some applications to
provide a source for reagents, which are supplied to the biochip when
pressure is applied to accumulator 26. In applications in which reagents
are not utilized, inlets 28 and reagent side accumulator 26 may not be
used. Additionally, two inlets 30 are provided in the exemplary
embodiment illustrated in FIG. 3 to provide hydration to the biochip.
Hydration inlets 30 are in fluid communication with the device to
facilitate the control of humidity associated with the reaction chambers.
As will be understood to one of skill in the art, some elastomeric
materials utilized in the fabrication of the device are gas permeable,
allowing evaporated gases or vapor from the reaction chambers to pass
through the elastomeric material into the surrounding atmosphere. In a
particular embodiment, fluid lines located at peripheral portions of the
device provide a shield of hydration liquid, for example, a buffer or
master mix, at peripheral portions of the biochip surrounding the panels
of reaction chambers, thus reducing or preventing evaporation of liquids
present in the reaction chambers. Thus, humidity at peripheral portions
of the device can be increased by adding a volatile liquid, for example
water, to hydration inlets 30. In a specific embodiment, a first inlet is
in fluid communication with the hydration fluid lines surrounding the
panels on a first side of the biochip and the second inlet is in fluid
communication with the hydration fluid lines surrounding the panels on
the other side of the biochip.

[0091] While the devices and sample distribution described above is one
exemplary system for carrying out the methods of the present invention,
one of ordinary skill in the art would recognize many variations,
modifications, and alternatives to designing the microfluidic devices
described herein. For example, although the microfluidic device
illustrated in FIG. 3 includes 12 panels, each having 765 reaction
chambers with a volume of 6 nL per reaction chamber, this is not required
by the present invention. The particular geometry of the digital array
will depend on the particular applications. Thus, e.g., the scope of the
present invention is not limited to digital arrays with 12 panels having
765 reaction chambers, but other combinations are included within the
scope of the present invention. Additional description related to digital
arrays suitable for use in embodiments of the present invention are
provided in U.S. Patent Application Publication No. 2005/0252773,
incorporated herein by reference.

[0092] Running large numbers of replicate samples can require significant
quantities of reagents. In an embodiment of the present invention,
digital PCR is conducted in microvolumes. The reaction chambers for
running low volume PCR may be from about 2 nL to about 500 nL. The lower
the reaction chamber volume, the more the number of individual assays
that may be run (either using different probe and primer sets or as
replicates of the same probe and primer sets or any permutation of
numbers of replicates and numbers of different assays). In one
embodiment, the reaction chamber is from about 2 nL to about 50 nL,
preferably 2 nL to about 25 nL, more preferably from about 4 nL to about
15 nL. In some embodiments, the reaction chamber volume is about 4 nL,
about 5 nL, about 6, nL, about 7 nL, about 8, nL, about 9 nL, about 10
nL, about 11 nL, or about 12, nL. The sample chambers may be constructed
of glass, plastic, silicon, elastomeric polymers such as
polydimethylsiloxane, polyurethane, or other polymers. The samples
processed by the method of the invention are well suited for use in
variable copy number analysis using the BioMark® system (Fluidigm
Corporation, South San Francisco, Calif.). The BioMark® system uses a
polydimethylsiloxane microfluidic device that provides for running
multiple assays on multiple samples.

[0093] The Fluidigm microfluidic devices (digital arrays) are manufactured
by Fluidigm Corporation (South San Francisco, Calif.). Chips are
fabricated following the Multilayer Soft Lithography (MSL) methodology
(Unger M A, Chou H P, Thorsen T, Scherer A, Quake S R, Monolithic
microfabricated valves and pumps by multilayer soft lithography, Science
2000; 288:113-116). The chip has sample channels that have 10 μm
average semi-elliptical depth, 70 μm width, with parallel spacing 200
μm on-center. Sample fluidics are fabricated with a two-layer mold
process to create partition chambers 265 μm (depth)×150
μm×150 μm arranged along each sample channel. On a separate
silicone layer, the control channels of the chip run perpendicular to the
sample channels. The intersections of the channels form deflective valves
for routing fluids. Upon pressurization of the control channels, a thin
membrane between layers closes off the sample channels to isolate
individual partition chambers. The control channels are 15 μm deep, 50
μm wide with parallel spacing 300 μm on center.

[0094] Reaction mixes, such as PCR mixes, sample mixes, pre-amplification
product sample mixes, are loaded into each panel and single DNA molecules
are randomly partitioned into the various reaction chambers. After
loading of the panels and reaction chambers, the digital array can be
thermocycled and then imaged on an appropriate reader, for example, a
BioMark® instrument available from the present assignee. The data
produced is analyzed using Digital PCR Analysis software available from
the present assignee or other suitable analysis software. Additional
description of exemplary detection and/or analysis techniques suitable
for use in embodiments of the present invention are provided in U.S.
patent application Publication No. U.S. application Ser. No. 12/170,414
entitled "Copy Number Variation Determination by Digital PCR," which is
copending and commonly assigned and hereby incorporated by reference for
all purposes.

[0095] FIGS. 4A-4C are simplified diagrams of portion of the
device/biochip illustrated in FIG. 3. FIG. 4A illustrates the 12 panels
22, each of the panels including a number of reaction chambers. FIG. 4B
illustrates the geometry of a number of reaction chambers 40 contained in
a panel. The reaction chambers 40 are spaced on 200 μm centers as
illustrated. FIG. 4c illustrates a fluorescence image of a portion of a
panel. The left side of the illustration is a control section, with all
the reaction chambers illustrated as dark. The right side of the
illustration shows how in a typical experiment, many of the reaction
chambers are dark 42, generating no significant fluorescent emission.
However, a portion of the reaction chambers have fluorescent emission,
indicating a "positive" reaction chamber 44. As described above in FIG.
2B, sample channels run left to right connecting individual reaction
chambers and control channels run top to bottom in the lower layer. Upon
pressurization of the control channels, a thin membrane between layers
closes off the sample channels to isolate individual reaction chambers.
The valves partition individual chambers that are kept closed during the
PCR experiment.

[0096] As described more fully throughout the present specification, the
chip was thermocycled and imaged on the BioMark® real-time PCR system
available from the present assignee and Digital PCR Analysis software,
such as the BioMark® Digital PCR Analysis available from the present
assignee, was used to count the number of positive chambers in each
panel. When two assays with two fluorescent dyes are used in a multiplex
digital PCR reaction, two genes can be independently quantitated. This
ability to independently quantitate genes is used as described herein to
study copy number variations using the digital array. The number of genes
that can be independently quantitated in a single PCR reaction is
dependent on the number of fluorescent dyes and filters available.

[0097] As described in the general methods steps above, following
distribution of the sample additional steps include an amplification step
followed by detection and analysis of results. In some embodiments of the
present invention, amplification and detection/analysis can be conducted
using methods that coordinate the two steps together, e.g., quantitative
PCR. Generally, polynucleotides that are isolated within each reaction
site can be amplified, detected and analyzed using a range of possible
strategies. One exemplary strategy involves amplifying target and
reference polynucleotides so that the amplified product can be used to
determine a concentration of target polynucleotide and a concentration of
the reference polynucleotide. To conduct the amplification, reagents
necessary for amplification are combined with the sample and can include
a first probe that selectively hybridizes to a target polynucleotide and
a second probe that selectively hybridizes to a reference polynucleotide
under conditions that are suitable for polynucleotide amplification. The
first and second probes can include different detectable labels, so as to
differentiate between the target and reference polynucleotide
amplification products. Furthermore, differentiation of the target and
reference polynucleotides can provide for further calculation of the
concentration of target nucleotide molecules as a ratio of the reference
nucleotide molecules so as to determine the relative copy number of the
target polynucleotide sequence in the genome of the subject.

[0098] The general steps of amplification followed by detection and
analysis can be performed using a number of ways.

[0099] To enhance understanding of the methods and systems described
throughout the specification, terms of art are generally described below.
The term "reagent" refers broadly to any agent used in a reaction. A
reagent can include a single agent which itself can be monitored (e.g., a
substance that is monitored as it is heated) or a mixture of two or more
agents. A reagent may be living (e.g., a cell) or non-living. Exemplary
reagents for a nucleic acid amplification reaction include, but are not
limited to, buffer, metal ions, polymerase, primers, template nucleic
acid, nucleotides, labels, dyes, nucleases and the like. Reagents for
enzyme reactions include, for example, substrates, cofactors, coupling
enzymes, buffer, metal ions, inhibitors and activators. Reagents for
cell-based reactions include, but are not limited to, cells, cell
specific dyes and ligands (e.g., agonists and antagonists) that bind to
cellular receptors. Reagents can be included in the sample solution, or
can optionally be immobilized in a variety of ways (e.g., covalently,
non-covalently, via suitable linker molecules). In on-chip nucleic acid
amplification reactions, for example, one or more reagents used in
conducting extension reactions can be deposited (e.g., through spotting)
at each of the reaction sites during manufacture of the device.

[0100] The term "label" refers to a molecule or an aspect of a molecule
that can be detected by physical, chemical, electromagnetic and other
related analytical techniques. Examples of detectable labels that can be
utilized include, but are not limited to, radioisotopes, fluorophores,
chromophores, mass labels, electron dense particles, magnetic particles,
spin labels, molecules that emit chemiluminescence, electrochemically
active molecules, enzymes, cofactors, enzymes linked to nucleic acid
probes and enzyme substrates. The term "detectably labeled" means that an
agent has been conjugated with a label or that an agent has some inherent
characteristic (e.g., size, shape or color) that allows it to be detected
without having to be conjugated to a separate label.

[0101] The terms "nucleic acid," "polynucleotide," and "oligonucleotide"
are used herein to include a polymeric form of nucleotides of any length,
including, but not limited to, ribonucleotides or deoxyribonucleotides.
There is no intended distinction in length between these terms. Further,
these terms refer only to the primary structure of the molecule. Thus, in
certain embodiments these terms can include triple-, double- and
single-stranded DNA, as well as triple-, double- and single-stranded RNA.
They also include modifications, such as by methylation and/or by
capping, and unmodified forms of the polynucleotide. More particularly,
the terms "nucleic acid," "polynucleotide," and "oligonucleotide,"
include polydeoxyribonucleotides (containing 2-deoxy-D-ribose),
polyribonucleotides (containing D-ribose), any other type of
polynucleotide which is an N- or C-glycoside of a purine or pyrimidine
base, and other polymers containing normucleotidic backbones, for
example, polyamide (e.g., peptide nucleic acids (PNAs)) and
polymorpholino (commercially available from the Anti-Virals, Inc.,
Corvallis, Oreg., as Neugene) polymers, and other synthetic
sequence-specific nucleic acid polymers providing that the polymers
contain nucleobases in a configuration which allows for base pairing and
base stacking, such as is found in DNA and RNA.

[0102] A "probe" is an nucleic acid capable of binding to a target nucleic
acid of complementary sequence through one or more types of chemical
bonds, usually through complementary base pairing, usually through
hydrogen bond formation, thus forming a duplex structure. The probe binds
or hybridizes to a "probe binding site." The probe can be labeled with a
detectable label to permit facile detection of the probe, particularly
once the probe has hybridized to its complementary target. The label
attached to the probe can include any of a variety of different labels
known in the art that can be detected by chemical or physical means, for
example. Suitable labels that can be attached to probes include, but are
not limited to, radioisotopes, fluorophores, chromophores, mass labels,
electron dense particles, magnetic particles, spin labels, molecules that
emit chemiluminescence, electrochemically active molecules, enzymes,
cofactors, and enzyme substrates. Probes can vary significantly in size.
Some probes are relatively short. Generally, probes are at least 7 to 15
nucleotides in length. Other probes are at least 20, 30 or 40 nucleotides
long. Still other probes are somewhat longer, being at least 50, 60, 70,
80, 90 nucleotides long. Yet other probes are longer still, and are at
least 100, 150, 200 or more nucleotides long. Probes can be of any
specific length that falls within the foregoing ranges as well.

[0103] A "primer" is a single-stranded polynucleotide capable of acting as
a point of initiation of template-directed DNA synthesis under
appropriate conditions (i.e., in the presence of four different
nucleoside triphosphates and an agent for polymerization, such as, DNA or
RNA polymerase or reverse transcriptase) in an appropriate buffer and at
a suitable temperature. The appropriate length of a primer depends on the
intended use of the primer but typically is at least 7 nucleotides long
and, more typically range from 10 to 30 nucleotides in length. Other
primers can be somewhat longer such as 30 to 50 nucleotides long. Short
primer molecules generally require cooler temperatures to form
sufficiently stable hybrid complexes with the template. A primer need not
reflect the exact sequence of the template but must be sufficiently
complementary to hybridize with a template. The term "primer site" or
"primer binding site" refers to the segment of the target DNA to which a
primer hybridizes. The term "primer pair" means a set of primers
including a 5' "upstream primer" that hybridizes with the complement of
the 5' end of the DNA sequence to be amplified and a 3' "downstream
primer" that hybridizes with the 3' end of the sequence to be amplified.

[0104] A primer that is "perfectly complementary" has a sequence fully
complementary across the entire length of the primer and has no
mismatches. The primer is typically perfectly complementary to a portion
(subsequence) of a target sequence. A "mismatch" refers to a site at
which the nucleotide in the primer and the nucleotide in the target
nucleic acid with which it is aligned are not complementary. The term
"substantially complementary" when used in reference to a primer means
that a primer is not perfectly complementary to its target sequence;
instead, the primer is only sufficiently complementary to hybridize
selectively to its respective strand at the desired primer-binding site.

[0105] The term "complementary" means that one nucleic acid is identical
to, or hybridizes selectively to, another nucleic acid molecule.
Selectivity of hybridization exists when hybridization occurs that is
more selective than total lack of specificity. Typically, selective
hybridization will occur when there is at least about 55% identity over a
stretch of at least 14-25 nucleotides, preferably at least 65%, more
preferably at least 75%, and most preferably at least 90%. Preferably,
one nucleic acid hybridizes specifically to the other nucleic acid. See
M. Kanehisa, Nucleic Acids Res. 12:203 (1984).

[0106] Detection occurs at a "detection section," or "detection region."
These terms and other related terms refer to the portion of the
microfluidic device at which detection occurs. As indicated above, with
devices utilizing certain designs (e.g., open channel design, blind
channel design, etc.), the detection section is generally the reaction
site as isolated by the valve associated with each reaction site. The
detection section for matrix-based devices is usually within regions of
flow channels that are adjacent an intersection, the intersection itself,
or a region that encompasses the intersection and a surrounding region.

[0107] As discussed above, exemplary copy number variation analyses can be
conducted using quantitative PCR methods on-chip. In particular,
quantitative PCR can involve both amplification of polynucleotides and
detection/analysis of the amplified products. In addition to qPCR, a
variety of so-called "real time amplification" methods or "real time
quantitative PCR" methods can also be utilized to determine the quantity
of a target nucleic acid present in a sample by measuring the amount of
amplification product formed during or after the amplification process
itself. Fluorogenic nuclease assays are one specific example of a real
time quantitation method which can be used successfully with the devices
described herein. This method of monitoring the formation of
amplification product involves the continuous measurement of PCR product
accumulation using a dual-labeled fluorogenic oligonucleotide probe--an
approach frequently referred to in the literature as the "TaqMan" method.

[0108] The probe used in such assays is typically a short (e.g., about
20-25 bases) polynucleotide that is labeled with two different
fluorescent dyes. The 5' terminus of the probe is typically attached to a
reporter dye and the 3' terminus is attached to a quenching dye, although
the dyes can be attached at other locations on the probe as well. The
probe is designed to have at least substantial sequence complementarity
with the probe binding site on the target nucleic acid. Upstream and
downstream PCR primers that bind to regions that flank the probe binding
site are also included in the reaction mixture.

[0109] When the probe is intact, energy transfer between the two
fluorophores occurs and the quencher quenches emission from the reporter.
During the extension phase of PCR, the probe is cleaved by the 5'
nuclease activity of a nucleic acid polymerase such as Taq polymerase,
thereby releasing the reporter from the polynucleotide-quencher and
resulting in an increase of reporter emission intensity which can be
measured by an appropriate detector.

[0110] One detector which is specifically adapted for measuring
fluorescence emissions such as those created during a fluorogenic assay
is the ABI 7700 manufactured by Applied Biosystems, Inc. in Foster City,
Calif. Computer software provided with the instrument is capable of
recording the fluorescence intensity of reporter and quencher over the
course of the amplification. These recorded values can then be used to
calculate the increase in normalized reporter emission intensity on a
continuous basis and ultimately quantify the amount of the mRNA being
amplified.

[0111] Additional details regarding the theory and operation of
fluorogenic methods for making real time determinations of the
concentration of amplification products are described, for example, in
U.S. Pat. Nos. 5,210,015 to Gelfand, 5,538,848 to Livak, et al., and
5,863,736 to Haaland, as well as Heid, C. A., et al., Genome Research,
6:986-994 (1996); Gibson, U. E. M, et al., Genome Research 6:995-1001
(1996); Holland, P. M., et al., Proc. Natl. Acad. Sci. USA 88:7276-7280,
(1991); and Livak, K. J., et al., PCR Methods and Applications 357-362
(1995), each of which is incorporated by reference in its entirety. Thus,
as the amplification reaction progresses, an increasing amount of dye
becomes bound and is accompanied by a concomitant increase in signal.

[0112] In performing amplification assays on-chip, multiplex
amplifications can be performed within a single reaction site by, for
example, utilizing a plurality of primers, each specific for a particular
target nucleic acid of interest (e.g., target polynucleotide sequence and
reference polynucleotide sequence), during the thermal cycling process.
The presence of the different amplified products can be detected using
differentially labeled probes to conduct a quantitative RT-PCR reaction
or by using differentially labeled molecular beacons (see supra). In such
approaches, each differentially labeled probes is designed to hybridize
only to a particular amplified target. By judicious choice of the
different labels that are utilized, analyses can be conducted in which
the different labels are excited and/or detected at different wavelengths
in a single reaction. Further guidance regarding the selection of
appropriate fluorescent labels that are suitable in such approaches
include: Fluorescence Spectroscopy (Pesce et al., Eds.) Marcel Dekker,
New York, (1971); White et al., Fluorescence Analysis: A Practical
Approach, Marcel Dekker, New York, (1970); Berlman, Handbook of
Fluorescence Spectra of Aromatic Molecules, 2nd ed., Academic Press,
New York, (1971); Griffiths, Colour and Constitution of Organic
Molecules, Academic Press, New York, (1976); Indicators (Bishop, Ed.).
Pergamon Press, Oxford, 19723; and Haugland, Handbook of Fluorescent
Probes and Research Chemicals, Molecular Probes, Eugene (1992).

[0113] When microfluidic devices such as open channel or blind channel
design devices are utilized to perform nucleic acid amplification
reactions, the reagents that can be deposited within the reaction sites
are those reagents necessary to perform the desired type of amplification
reaction. Usually this means that some or all of the following are
deposited, primers, polymerase, nucleotides, metal ions, buffer, and
cofactors, for example. The sample introduced into the reaction site in
such cases is the nucleic acid template. Alternatively, however, the
template can be deposited and the amplification reagents flowed into the
reaction sites. When the matrix device is utilized to conduct an
amplification reaction, samples containing nucleic acid template can be
flowed through the vertical flow channels and the amplification reagents
through the horizontal flow channels or vice versa.

[0114] In general, multiple genotyping and expression analyses can be, for
example, conducted at each reaction site. Sample containing the target
DNA can be introduced into reaction sites on a microfluidic device. For
quantitative PCR methods such as TaqMan®, primers for amplifying
different regions of a target DNA of interest are included within a
single reaction site. Differentially labeled probes for each region are
utilized to distinguish product that is formed, e.g. target and reference
polynucleotides. If the allele to which a probe is complementary is
present in the target DNA, then amplification occurs, thereby resulting
in a detectable signal. Based upon which of the differential signal is
obtained, the identity of the nucleotide at the polymorphic site can be
determined. If both signals are detected, then both alleles are present.
Thermocycling during the reaction is performed as described in the
temperature control section supra.

[0115] In some embodiments of the present invention, differentially
labeled probes complementary to each of the allelic forms can be included
as reagents, together with primers, nucleotides and polymerase. However,
reactions can be conducted with only a single probe, although this can
create ambiguity as to whether lack of signal is due to absence of a
particular allele or simply a failed reaction. For the typical biallelic
case in which two alleles are possible for a polymorphic site, two
differentially labeled probes, each perfectly complementary to one of the
alleles are usually included in the reagent mixture, together with
amplification primers, nucleotides and polymerase.

[0116] As indicated by FIG. 4c, signal from each reaction site can be
detected and further analyzed to determine information about the sample.
For example, the samples processed by the methods of the invention are
well suited for use in variable copy number analysis using the
BioMark® system (Fluidigm Corporation, South San Francisco, Calif.).
and BioMark® fluorescence imaging thermal cycler system. The
BioMark® system uses a polydimethylsiloxane microfluidic device that
provides for running multiple assays on multiple samples.

[0117] As described more fully throughout the present specification, the
chip can in some embodiments be thermocycled and imaged on the
BioMark® real-time PCR system available from the present assignee and
Digital PCR Analysis software, such as the BioMark® Digital PCR
Analysis available from the present assignee, was used to count the
number of positive chambers in each panel. When two assays with two
fluorescent dyes are used in a multiplex digital PCR reaction, two genes
can be independently quantitated. This ability to independently
quantitate genes is used as described herein to study copy number
variations using the digital array.

[0118] As described generally above, reaction mixes, such as PCR mixes,
can be loaded into each panel and single DNA molecules can be randomly
partitioned into the various reaction chambers. After loading of the
panels and reaction chambers, the digital array is thermocycled and then
imaged on an appropriate reader, for example, a BioMark® instrument
available from the present assignee. The data produced is analyzed using
Digital PCR Analysis software available from the present assignee or
other suitable analysis software.

[0119] As described above, quantitative PCR on-chip can be used to carry
out certain embodiments of the present invention. Though, a number of
different detection strategies can be utilized with the microfluidic
devices described above. Selection of the appropriate system is informed
in part on the type of device, event and/or agent being detected. The
detectors can be designed to detect a number of different signal types
including, but not limited to, signals from radioisotopes, fluorophores,
chromophores, electron dense particles, magnetic particles, spin labels,
molecules that emit chemiluminescence, electrochemically active
molecules, enzymes, cofactors, enzymes linked to nucleic acid probes and
enzyme substrates.

[0121] The detection section can be in communication with one or more
microscopes, diodes, light stimulating devices (e.g., lasers),
photomultiplier tubes, processors and combinations of the foregoing,
which cooperate to detect a signal associated with a particular event
and/or agent. Often the signal being detected is an optical signal that
is detected in the detection section by an optical detector. The optical
detector can include one or more photodiodes (e.g., avalanche
photodiodes), a fiber-optic light guide leading, for example, to a
photomultiplier tube, a microscope, and/or a video camera (e.g., a CCD
camera).

[0122] Detectors can be microfabricated within the microfluidic device, or
can be a separate element. If the detector exists as a separate element
and the microfluidic device includes a plurality of detection sections,
detection can occur within a single detection section at any given
moment. Alternatively, scanning systems can be used. For instance,
certain automated systems scan the light source relative to the
microfluidic device; other systems scan the emitted light over a
detector, or include a multichannel detector. As a specific illustrative
example, the microfluidic device can be attached to a translatable stage
and scanned under a microscope objective. A signal so acquired is then
routed to a processor for signal interpretation and processing. Arrays of
photomultiplier tubes can also be utilized. Additionally, optical systems
that have the capability of collecting signals from all the different
detection sections simultaneously while determining the signal from each
section can be utilized.

[0123] External detectors are usable because the devices that are provided
are completely or largely manufactured of materials that are optically
transparent at the wavelength being monitored. This feature enables the
devices described herein to utilize a number of optical detection systems
that are not possible with conventional silicon-based microfluidic
devices.

[0124] In one embodiment, a detector uses a CCD camera and an optical path
that provides for a large field of view and a high numerical aperture to
maximize the amount of light collected from each reaction chamber. In
this regard, the CCD is used as an array of photodetectors wherein each
pixel or group of pixels corresponds to a reaction chamber rather than
being used to produce an image of the array. Thus, the optics may be
altered such that image quality is reduced or defocused to increase the
depth of field of the optical system to collect more light from each
reaction chamber.

[0125] A detector can include a light source for stimulating a reporter
that generates a detectable signal. The type of light source utilized
depends in part on the nature of the reporter being activated. Suitable
light sources include, but are not limited to, lasers, laser diodes and
high intensity lamps. If a laser is utilized, the laser can be utilized
to scan across a set of detection sections or a single detection section.
Laser diodes can be microfabricated into the microfluidic device itself.
Alternatively, laser diodes can be fabricated into another device that is
placed adjacent to the microfluidic device being utilized to conduct a
thermal cycling reaction such that the laser light from the diode is
directed into the detection section.

[0126] Detection can involve a number of non-optical approaches as well.
For example, the detector can also include, for example, a temperature
sensor, a conductivity sensor, a potentiometric sensor (e.g., pH
electrode) and/or an amperometric sensor (e.g., to monitor oxidation and
reduction reactions).

[0127] A number of commercially-available external detectors can be
utilized. Many of these are fluorescent detectors because of the ease in
preparing fluorescently labeled reagents. Specific examples of detectors
that are available include, but are not limited to, Applied Precision
ArrayWoRx (Applied Precision, Issaquah, Wash.)).

[0128] In some embodiments FRET-based detection methods are used.
Detection methods of this type involve detecting a change in fluorescence
from a donor (reporter) and/or acceptor (quencher) fluorophore in a
donor/acceptor fluorophore pair. The donor and acceptor fluorophore pair
are selected such that the emission spectrum of the donor overlaps the
excitation spectrum of the acceptor. Thus, when the pair of fluorophores
are brought within sufficiently close proximity to one another, energy
transfer from the donor to the acceptor can occur. This energy transfer
can be detected. See U.S. Pat. No. 5,945,283 and PCT Publication WO
97/22719.

[0129] Molecular Beacons provide a particularly useful approach. With
molecular beacons, a change in conformation of the probe as it hybridizes
to a complementary region of the amplified product results in the
formation of a detectable signal. The probe itself includes two sections:
one section at the 5' end and the other section at the 3' end. These
sections flank the section of the probe that anneals to the probe binding
site and are complementary to one another. One end section is typically
attached to a reporter dye and the other end section is usually attached
to a quencher dye.

[0130] In solution, the two end sections can hybridize with each other to
form a hairpin loop. In this conformation, the reporter and quencher dye
are in sufficiently close proximity that fluorescence from the reporter
dye is effectively quenched by the quencher dye. Hybridized probe, in
contrast, results in a linearized conformation in which the extent of
quenching is decreased. Thus, by monitoring emission changes for the two
dyes, it is possible to indirectly monitor the formation of amplification
product. Probes of this type and methods of their use is described
further, for example, by Piatek, A. S., et al., Nat. Biotechnol.
16:359-63 (1998); Tyagi, S, and Kramer, F. R., Nature Biotechnology
14:303-308 (1996); and Tyagi, S. et al., Nat. Biotechnol. 16:49-53
(1998), each of which is incorporated by reference herein in their
entirety for all purposes.

[0132] As indicated above, methods of the present invention include
conducting various reactions/amplification assays that require various
reagents, compositions, buffers, additives, and the like. Reaction
mixtures can be prepared at least partially either separate from an assay
platform or microfluidic chip/device, or within reaction sites of the
device itself (e.g., spotting). Certain reaction mixtures or compositions
can be prepared and included as part of a kit or system. For example, a
system can include a pre-amplification mixture/composition, an
amplification assay composition, and a microfluidic device for performing
amplification and copy number detection assays. Two or more components of
the system can be assembled and provided as part of a kit or system.

[0133] Reactions conducted with the microfluidic devices disclosed herein
can be conducted with various reagents, buffers, compositions, additives,
and the like, which can be formulated to conduct reactions of the present
invention (e.g., pre-amplification, quantitative amplification, etc.).
So, for example, in the case of devices in which reagents are deposited
reagents can be spotted with one or more reactants at a reaction site,
for instance. In other embodiments, e.g., when on-chip spotting does not
occur, reagents can be provided in mixes or reagent volumes separate from
the chip or other system components. One set of additives are blocking
reagents that block protein binding sites on the elastomeric substrate. A
wide variety of such compounds can be utilized including a number of
different proteins (e.g., gelatin and various albumin proteins, such as
bovine serum albumin) and glycerol. A detergent additive can also be
useful. Any of a number of different detergents can be utilized. Examples
include, but are not limited to SDS and the various Triton detergents.

[0134] In the specific case of nucleic acid amplification reactions, a
number of different types of reagents and/or additives can be included.
One category are enhancers that promote the amplification reaction. Such
additives include, but are not limited to, reagents that reduce secondary
structure in the nucleic acid (e.g., betaine), and agents that reduce
mispriming events (e.g., tetramethylammonium chloride).

[0135] Generally, the CNV calculation can be based on "relative copy
number" so that apparent differences in gene copy numbers in different
samples are not distorted by differences in sample amounts. The relative
copy number of a gene (per genome) can be expressed as the ratio of the
copy number of a target gene to the copy number of a single copy
reference gene in a DNA sample, which is typically 1. By using two assays
for the two genes (the target polynucleotide sequence and the reference
polynucleotide sequence) with two different fluorescent dyes on the same
device, both genes in the same DNA sample can be quantitated
simultaneously. Accordingly, the ratio of the two genes is the relative
copy number of the target nucleotide sequence in a DNA sample.

[0136] In one embodiment of the present invention, pre-amplification can
be conducted using a reference gene such as RNaseP which is a single-copy
gene that encodes the RNA moiety for the RNaseP enzyme, a
ribonucleoprotein.

[0137] Running large numbers of replicate samples can require significant
quantities of reagents. In an embodiment of the present invention,
digital PCR is conducted in microvolumes. The reaction chambers for
running low volume PCR may be from about 2 nL to about 500 nL. The lower
the reaction chamber volume, the more the number of individual assays
that may be run (either using different probe and primer sets or as
replicates of the same probe and primer sets or any permutation of
numbers of replicates and numbers of different assays). In one
embodiment, the reaction chamber is from about 2 nL to about 50 nL,
preferably 2 nL to about 25 nL, more preferably from about 4 nL to about
15 nL. In some embodiments, the reaction chamber volume is about 4 nL,
about 5 nL, about 6, nL, about 7 nL, about 8, nL, about 9 nL, about 10
nL, about 11 nL, or about 12, nL. The sample chambers may be constructed
of glass, plastic, silicon, elastomeric polymers such as
polydimethylsiloxane, polyurethane, or other polymers. The samples
processed by the methods of the present invention are well suited for use
in variable copy number analysis using the BioMark® system (Fluidigm
Corporation, South San Francisco, Calif.). The BioMark system uses a
polydimethylsiloxane microfluidic device that provides for running
multiple assays on multiple samples.

[0138] The Fluidigm devices/nanofluidic chips (digital arrays) and BioMark
fluorescence imaging thermal cycler system are manufactured by Fluidigm
Corporation (South San Francisco, Calif.). An exemplary chip as
illustrated in FIG. 5 has 12 panels and each of the 12 panels contains
765 6-nL chambers with a total volume of 4.59 μL per panel. Chips are
fabricated following the Multilayer Soft Lithography (MSL) methodology.
Unger M A, Chou H P, Thorsen T, Scherer A, Quake SR. Monolithic
microfabricated valves and pumps by multilayer soft lithography. Science.
2000; 288:113-116. The chip has sample channels that have 10 μm
average semi-elliptical depth, 70 μm width, with parallel spacing 200
μm on-center. Sample fluidics are fabricated with a two-layer mold
process to create partition chambers 265 μm (depth)×150
μm×150 μm arranged along each sample channel. On a separate
silicone layer, the control channels of the chip run perpendicular to the
sample channels. The intersections of the channels form deflective valves
for routing fluids. Upon pressurization of the control channels, a thin
membrane between layers closes off the sample channels to isolate
individual partition chambers. The control channels are 15 μm deep, 50
μm wide with parallel spacing 300 μm on center. The outer portion
has the same footprint as a standard 384-well microplate and enables
stand-alone valve operation. There are 12 input ports corresponding to 12
separate sample inputs to the chip. The chips used can incorporate 765 6
nL partitioning chambers per sample input, for a total of up to 14,400
chambers per chip. In this particular embodiment, sample channels run
left to right connecting individual reaction chambers and control
channels run top to bottom in the lower layer. Upon pressurization of the
control channels, a thin membrane between layers closes off the sample
channels to isolate individual reaction chambers. The valves partition
individual chambers that are kept closed during the PCR experiment.

[0139] For running real time PCR reactions, a master amplification mix
(e.g., "master mix") is combined with sample including product of the
pre-amplification assay. Master mixes contain an appropriate buffer, a
source of magnesium ions (Mg2+) in the range of about 1 to about 10 mM,
preferably in the range of about 2 to about 8 mM, nucleotides, and
optionally, detergents, and stabilizers. An example of one suitable
buffer is TRIS buffer at a concentration of about 5 mM to about 85 mM,
with a concentration of 10 mM to 30 mM preferred. In one embodiment, the
TRIS buffer concentration is 20 mM in the reaction mix double strength
(2×) form. The reaction mix can have a pH range of from about 7.5
to about 9.0, with a pH range of about 8.0 to about 8.5 as typical.
Concentration of nucleotides can be in the range of about 25 mM to about
1000 mM, typically in the range of about 100 mM to about 800 mM. Examples
of dNTP concentrations are 100, 200, 300, 400, 500, 600, 700, and 800 mM.
Detergents such as Tween® 20, Triton® X 100, and Nonidet® P40
may also be included in the reaction mixture. Stabilizing agents such as
dithiothreitol (DTT, Cleland's reagent) or mercaptoethanol may also be
included.

[0140] DO WE NEED THIS PARAGRAPH? In addition, master mixes may optionally
contain dUTP as well as uracil DNA glycosylase (uracil-N-glycosylase,
UNG). UNO is the product of the Escherichia coli ung gene, and has been
cloned, sequenced and expressed in E. coli. Uracil-DNA-N-glycosylase
(UNG) removes uracil residues from DNA (single- and double stranded)
without destroying the DNA sugar-phosphodiester backbone; thus,
preventing its use as a hybridization target or as a template for DNA
polymerases. The resulting abasic sites are susceptible to hydrolytic
cleavage at elevated temperatures. Thus, removal of uracil bases is
usually accompanied by fragmentation of the DNA. Duncan, B. K., and
Chambers, J. A. (1984) GENE 28, 211, Varshney, U., Hutcheon, T., and van
de Sande, J. H. (1988) 1. Biol. Chem. 263, 7776. A master mix is
commercially available from Applied Biosystems, Foster City, Calif.,
(TaqMan® Universal Master Mix, cat. nos. 4304437, 4318157, and
4326708). The use of UNG will typically be restricted to the digital PCR
assay and not used in the pre-amplification assay.

[0141] For multiplex applications, different fluorescent reporter dyes are
used to label separate primers or probes for quantification of different
genes. For relative expression studies using multiplex PCR, the amount of
primer for the reference gene (e.g., β-actin or GAPDH) should be
limited to avoid competition between amplification of the reference and
the sample gene. In general, the final concentration of the reference
gene primer should be between 25 and 100 nM. A primer titration can be
useful for optimization.

Example

[0142] In one exemplary embodiment of the present invention, the copy
number of CYP2D6 was determined with and without pre-amplification. Using
pre-amplification, the CYP2D6 in one sample was discovered to have a
duplication (copy number was 3), whereas without pre-amplification the
same sample showed a copy number of 2.

[0144] Pre-amplification in one example was performed on GeneAmp PCR
system 9700 (Applied Biosystems, CA) in a 5 μL reaction containing
I× PreAmp master mix (Applied Biosystems, CA), 225 nM primers
(RNase P as the reference polynucleotide) and the target sequence of
interest), and 1 μL of DNA sample. Thermal cycling conditions were
95° C., 10 minute hot start and 10 cycles of 95° C. for 15
seconds and 60° C. for 1 minute. 20 μL of water is added to
each reaction after pre-amplification and the samples were analyzed on
the digital array.

[0145] Five Coriell DNA samples were analyzed on the digital chips. The
numbers of the CYP2D6 and RNase P molecules in the same volume (4.59
μL) of each sample were counted by using the BioMark Digital PCR
Analysis software using the Poisson correction as well as Simant's
algorithm (see Dube et al., supra.) A representative heat map is shown in
simplified black and white illustration in FIG. 5. While shown as white,
black, and gray events for illustration purposes, events can be recorded
and graphically displayed as colors such as yellow, green, or red,
corresponded to an RNase P gene (VIC, yellow), a CYP2D6 gene (FAM, red),
and no gene, respectively. No template controls (NTC) were run in panels
1 and 12.

[0146] The ratios of the numbers of molecules of the CYP2D6 gene to the
RNase P gene were obtained for the five samples. Two of the ratios were
about 0.5, meaning there is only one copy of the CYP2D6 gene in each cell
of these two samples (RNase P is a single copy gene and there are always
two copies of the gene in each cell). Therefore, the individuals from
which the DNA samples were collected must have a deletion of the CYP2D6
gene on one chromosome. The other three samples had a ratio of about 1,
but this does not rule out the possibility of duplication since two
closely linked copies will be on one molecule and can not be separated. A
pre-amplification reaction was performed on these five samples and the
preamp products were analyzed on the digital chips (Table 2).

[0147] As illustrated in Table 2, two samples with a CYP2D6 to RNase P
ratio of about 0.5 when genomic DNA was used still gave a ratio of about
0.5 when the preamplification process of the invention was used. A 0.5
ratio indicates a deletion. Two samples with a ratio of about 1 when
genomic DNA was used also had a ratio of about 1 with preamplification
products, which indicated a normal allelic status. But, one sample with a
ratio of about 1 when genomic DNA was analyzed had a ratio of 1.5 when
the preamplification process was used. This indicates that the sample has
a duplication of the CYP2D6 gene.

[0148] Detecting Loss of Heterozygosity

[0149] One useful application of the described methods of determining copy
number variation of a particular gene of interest includes detecting a
loss of heterozygosity (LOH). The techniques disclosed herein can offer a
new level of sensitivity and flexibility in detecting loss of
heterozygosity. Exemplary applications include detection and/or study
abnormal X chromosome copy number, or aneuploidy. Loss of heterozygosity
(LOH) refers to a change from a heterozygous state in a normal genome to
a homozygous state in a paired tumor genome. Research shows that the loss
of an entire X chromosome is involved in numerous cancers. Moertel, C. A.
et al., Cancer Genet. Cytogenet. 67:21-27 (1993). For example, 40 percent
of ovarian cancers are associated with LOH for regions of the X
chromosome. Osbourne, R. J. and Leech, V., Br. J. Cancer 69:429-438
(1994). Also, the gain of an X chromosome has been shown to be relatively
common in leukemias and lymphomas. Sandberg A A. "The X chromosome in
human neoplasia, including sex chromatin and congenital conditions with
X-chromosome anomalies. In: Sandberg A A, editor. Cytogenetics of the
mammalian X chromosome, part B: X chromosome anomalies and their clinical
manifestations. New York: Alan R. Liss, 459-98 (1983).

[0150] To carry out LOH experiments, microfluidic devices as described
herein can be provided. FIG. 3 shows the architecture of an exemplary
device that was used for determining loss of heterozygosity in one
example (see, e.g., above discussion for more device detail). Briefly,
the device includes an integrated fluidic circuit (IFC) having 12 panels,
each having a flow input for a sample or assay mixture. In one example,
the sample was transferred to the chip for loading, and loaded by placing
the digital array on the IFC controller and using the software interface
to pressure load the assay components into separate panels of 765
reactions. Each of the twelve samples, which were premixed with master
mix and primer-probe sets, were distributed into separate inlets on the
frame of the chip. Within each panel, a single sample was partitioned
into 765 individual 6 nL real-time PCR reactions. PCR was performed with
the sample. The digital array was placed on a real-time PCR system for
thermal cycling and fluorescence detection. The results from the
experiment were viewed and analyzed using BioMark® application
software. Real-time PCR curves or end point images of positive chambers
were recorded to compare one assay versus another assay, e.g., the ratio
of any two sequences in a DNA sample were calculated. For analysis, the
digital arrays offer improved linearity, sensitivity, and ease of use.

[0151] In the described example, DNA from cell lines containing 1, 2, 3, 4
or 5 copies of the X chromosome (Coriell Institute for Medical Research,
Camden, N.J.) were obtained. Digital arrays were used to test each sample
against three separate X chromosome TaqMan® primer-probe
sets--FAM-labeled 123B, SMS, and YY2 (BioSearch Technologies, Novato,
Calif.)--which were co-amplified in the presence of a
single-copy-targeting, VIC-labeled "reference" sequence.

[0152]FIG. 6 shows a black and white diagram illustrating a color-based
results examining loss of heterozygosity as described, and further
illustrates each test run in duplicate panels within digital arrays. FIG.
6 also shows an up-close view of Panel 4 of the device. In each panel,
the number of Target positive (light gray, which correspond to one color,
e.g., yellow) and Reference positive (darker gray, which correspond to a
second color, e.g., red) chambers were counted and corrected for multiple
dyes per chamber. From these results, the raw ratio of Target to
Reference was determined. No template controls (NTC) were used in panels
1 and 12. It will be appreciated that in practice experiments can record
different colors and results illustrated in color, such as red and
yellow, which are depicted in FIG. 6 as grays in the black and white
illustration.

[0153] Simple linear fitting was used to determine copy numbers. FIG. 7
shows the average of three separate assay ratios (Y-axis) plotted against
known X chromosome copy number (X-axis), including error bars that show
the standard error of the mean. The ratios produced slopes for DNA
samples known to contain 1, 2, 3, 4 or 5 copies of the X chromosome. The
individual raw ratio measurements were multiplied by 2 and averaged to
obtain copy number per diploid genome. The average response for all
assays, over 1-to-5 copy number variants, was an r2 value of 0.994,
indicating high linear assay performance.

[0154] Table 3 lists the raw ratios from the TaqMan® primer probe sets
for individual X chromosome tests run on the microfluidic devices. The X
chromosome mean copies per genome was determined by multiplying the mean
ratio by 2. The last column on the right shows the standard error of the
mean (SEM). As shown in Table 3, the mean copies per genome corresponded
well with the known X chromosome copy number of a sample.

[0155] These results illustrate that methods and devices described herein
allow detection and distinguishing of small, yet biologically relevant,
differences in gene copy number within highly complex genomic DNA
samples. The samples selected for these tests are similar or identical to
those examined in CGH assays and MIP-based microarrays studies as
described in Visakorpi et al., 1994, Am. J. Pathol., 145:624-630 and
Pinkel et al., 1998, Nat. Genet. 20:207-211. The present results using
the methods of the current invention with digital arrays can produce copy
number estimations at least as discriminating as known CGH and MIP
methods while reducing hands-on technical manipulation and, therefore,
requiring less labor and increased efficiency. Moreover, the ability to
run multiple TaqMan® assays in a digital PCR format provides both
biological robustness and assay redundancy, compensating for
assay-to-assay amplification differences. If multiple loci are targeted
simultaneously, overall assay results are valid even if there are single
mutations or deletions at localized primer--probe binding sites.
Moreover, efficacy can be enhanced by using a pre-amplification step
prior to transferring the sample onto the microfluidic devices for
analysis.

[0156] Although the invention has been described with reference to the
above examples, it will be understood that modifications and variations
are encompassed within the spirit and scope of the invention.
Accordingly, the invention is limited only by the following claims along
with their full scope of equivalents.